Supplemental Table 1. Primers used for PCR.

Transcription

1 Supplemental Table 1. Primers used for PCR. Gene Type Primer Sequence Genotyping and semi-quantitative RT-PCR F 5 -TTG CCC GAT CAC CAT CTG TA-3 rwa1-1 R 5 -TGT AGC GAT CAA GGC CTG ATC TAA-3 LB 5 -TAG CAT CTG AAT TTC ATA ACC AAT CTC GAT ACA C-3 F 5 -CAC CCT GAA TTT CAT CTC AGC-3 rwa2-1 R 5 -TTG AAT AGG CAT CAA ACC GAG-3 LB 5 -ATA TTG ACC ATC ATA CTC ATT GC-3 F 5 -TCG AAT AGT CCA CTC TGG TCC-3 rwa2-3 R 5 -GCC TCT TGC ATA TTG TTC TTC C-3 LB 5 -ATT TTG CCG ATT TCG GAA C-3 F 5 -CAG CCT CTT GTC GTC TTT TGT TG-3 rwa3-1 R 5 -TGT GAT GCC AAG AGA AAG ATG TC-3 LB 5 -ATT TTG CCG ATT TCG GAA C-3 F 5 -ACA CGA ATG GCA TTT CAG ATC-3 rwa4-1 R 5 -ATA GCA ATC ACG TTG TGG AGC-3 LB 5 -TAG CAT CTG AAT TTC ATA ACC AAT CTC GAT ACA C-3 F 5 -GGG CTA AAG GAC ACT ACA CTG-3 ß-Tubulin R 5 -CCT CCT GCA CTT CCA CTT CGT CTT C-3 Quantitative RT-PCR F 5 -TCG GAA TTT CAC CCA CCA GCT TC-3 RWA1 R 5 -CCC GTC AGG CAT GTT TGA TCT TAG-3 F 5 -ATC GGA AAT GGC GAG TTC AAG CC-3 RWA2 R 5 -ATC CCG AAC ACC ACC GAC ATT AG-3 F 5 -ACG GCC ATC TAT GTC TTG GTG TC-3 RWA3 R 5 -ATT GTG GAG CAG CCT CTT GTC G-3 F 5 -CAC GGC CAT CTA CGT CTT GGT ATC-3 RWA4 R 5 -AGC AGC CGT TTG TCG TCT TTC G-3 F 5 -CTC AAA GGA ATG ATC TCG GAC ATA-3 PAD3 R 5 -TCT CCT TCT TGT CCC CAA GTG T-3 F 5 -CAC CCT TAT CTT CGC TGC TC-3 PDF1.2 R 5 -TGT TTG GCT CCT TCA AGG TT-3 F 5 -TAA CGT GGC CAA AAT GAT GC-3 PP2AA3 R 5 -GTT CTC CAC AAC CGC TTG GT-3 F 5 -CTG CGA CTC AGG GAA TCT TCT AA-3 UBC R 5 -TTG TGC CAT TGA ATT GAA CCC-3 F 5 -TCT TCC GCT CTT TCT TTC CAA GC-3 ACT2 R 5 -ACC ATT GTC ACA CAC GAT TGG TTG-3 Gateway cloning F 5 -CAG GCT TCA CCA TGG CGA GTT CAA GCC CTG TTA C-3 1 st step R 5 -AAA GCT GGG TCC ACC AGT TTT TGG GGA ATT GTG A-3 F 5 -GGG GAC AAG TTT GTA CAA AAA AGC AGG CTT CAC C-3 2 nd step R 5 -GGG GAC CAC TTT GTA CAA GAA AGC TGG GTC-3 Underlines indicate common regions of 1 st and 2 nd PCR primers for Gateway cloning.

2 Monosaccharide (mg/g dry weight) qrt1 rwa1-1 rwa2-1 rwa3-1 rwa4-1 Fuc Rha Ara Gal Glc Man Xyl GalA GlcA Supplemental Figure S1. There is no significant difference in cell wall monosaccharide composition between wildtype, rwa1-1, rwa2-1, rwa3-1, and rwa4-1. AIR was prepared from rosette leaves of short-day-grown mature plants. Then the AIR was destarched and hydrolyzed with TFA before being analyzed by highperformance anion exchange chromatography. Values shown are mean ± SE (n=3).

3 ph 7. ph 12.4 LM23 A rwa2 LM23 C ep cp P pp mx px LM2 E if LM1 G B D F H Supplemental Figure S2. Indirect immunofluorescence detection of the LM1, LM2 and LM23 epitopes in stem sections of wild-type () and rwa2-1. Transverse stem sections were pre-treated with phosphate buffer, ph 7. (A, C, E, G) or with.1 M sodium carbonate, ph 12.4 (B, D, F, H) and labeled with LM23 (A-D), LM2 (E and F) or LM1 (G and H). The LM23 antibody did not bind in the control sections and only the lignin autofluorescence was detected (A). In contrast, a weak labeling was detected at the epidermis in rwa2 (C). Epidermis labeling with LM23 is difficult to see at this magnification; pictures at higher magnification are shown in Figure 6. Alkaline pre-treatment strongly unmasked the LM23 epitope at the interfascicular fibers and xylem cells of wild-type and rwa2 (B and D). LM2 (which recognizes highly methylated homogalacturonan) bound to most cell types in the control sections (E), and this binding was lost after saponification (F) as observed in tobacco (Verhertbruggen et al., 29). The LM1 xylan antibody bound to interfascicular fibers in the control sections (G), and additionally to xylem cells after alkaline pre-treatment (H). Arrowheads show the outer surface of the epidermis. The dashed arrows show the metaxylem, which is labeled by LM1 after alkaline pre-treatment (G and H). ep: epidermis, cp: cortical parenchyma, P: phloem, if: interfascicular fibres, mx: metaxylem, px: protoxylem, pp: pith parenchyma. Scale bars: (A-H) 1 mm.

4 Monosaccharide (mg/g dry weight) Cell Wall Material Residue Supernatant rwa2-1 Fuc Rha Ara Gal Glc Man Xyl GalAGlcA Total Supplemental Figure S3. Monosaccharide composition analysis of wildtype and rwa2-1 cell wall before and after pectinase digestion. Cell Wall Material was prepared from rosette leaves of mature plants and treated with EPG and PME. Cell Wall Material prior to enzyme digestion, supernatant after digestion, and remaining pellet were hydrolyzed with TFA and analyzed by high-performance anion exchange chromatography. The sugar content in Cell Wall Material and Supernatant were calculated per g dry weight of Cell Wall Material. For the pellet, the sugars are calculated per g dry weight of pellet. There were no significant differences observed in monosaccharide composition. Values shown are mean ± SE (n=4 for rwa2-1, n=5 for ).

5 Supplemental Figure S4. Infection of wild-type and rwa2 mutant leaves with Golovinomyces cichoracearum.three-week-old plants were inoculated with spores from cucumber leaves infected 1 days prior to inoculation. Representative rosette leaves were photographed 12 days post inoculation. The experiment was repeated twice with similar results.

6 1. A PDF B PAD3 Relative expression.5 rwa2-1 rwa rwa2-1 rwa2-3 Supplemental Figure S5. Expression analysis of pathogen response genes. The expression of marker genes for pathogen response was tested in uninfected leaves of wild-type and rwa2 leaves. RNA was prepared using the RNeasy kit (Qiagen,Valencia, CA). The samples were treated with DNase using DNA-free TM (Applied Biosystems/Ambion, Austin, TX) before RNA (375 ng) was retrotranscribed into cdna (iscript cdna synthesis kit, Biorad, Hercules, CA). Real-time PCR was performed using DyNAmo TM Flash QPCR SYBR Green Mix (Finnzymes, Espoo, Finland) on a Rotor-Gene 6 (Qiagen, Valencia, CA). Gene expression values were normalized to expression of subunit A3 of protein phosphatase 2A (At1g1332). The primers used are listed in Supplemental Table 1.

7 Supplemental Figure S6. Multiple sequence alignment of Arabidopsis RWA proteins and the C-terminal domain of C. neoformans Cas1p. All four RWA proteins have more than one gene model, but they differ only in minor details. The protein sequences used for the alignment correspond to the default gene models according to Signal-Salk T- DNA Express ( Residues conserved in all five proteins are indicated with asterisks. The blue shading indicates transmembrane domains conserved in all 1TM acyltransferases, and the green letters indicate residues conserved in all 1TM acyltransferases and proposed to constitute an inter-membrane active site (Anantharaman and Aravind, 21). Yellow shading indicates additional transmembrane helices predicted in Cas1p and RWA proteins but not in all 1TM acyltransferases.